Clinical characteristics of colonization of the amniotic cavity in women with preterm prelabor rupture of membranes, a retrospective study

To determine the main clinical characteristics of preterm prelabor rupture of membranes (PPROM) complicated by colonization of the amniotic cavity (microbial invasion of the amniotic cavity without intra-amniotic inflammation). A total of 302 women with PPROM were included. Transabdominal amniocentesis was performed and amniotic fluid was assessed. Based of microbial invasion of the amniotic cavity and intra-amniotic inflammation (interleukin-6 ≥ 3000 pg/mL), the women were divided into following groups: intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid. Colonization was found in 11% (32/302) of the women. The most common bacteria identified in the amniotic fluid were Ureaplasma spp. with a lower burden than those with intra-amniotic infection (p = 0.03). The intensity of intra-amniotic inflammatory response measured by interleukin-6 was higher in women with colonization than in those with negative amniotic fluid (medians: 961 pg/mL vs. 616 pg/mL; p = 0.04). Women with colonization had higher rates of acute inflammatory placental lesions than those with negative amniotic fluid. In PPROM, colonization, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response.

Latency interval (hours) from rupture of membranes to delivery in women with preterm prelabor rupture of membranes with respect to intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid is shown in Fig. 3. No difference in the latency interval from rupture of membranes to delivery was found between colonization and negative amniotic fluid (colonization: median 76 h, IQR 30-132 vs. negative amniotic fluid: median 55 h, IQR 19-179; p = 0.76). However, women with colonization had a shorter latency interval than women with sterile intra-amniotic inflammation (median 146 h, IQR 37-428; p = 0.03). Comparisons with women with intra-amniotic infection were not made due to a different management approach (active management beyond the 28th week of gestation).
Short-term neonatal outcomes. The short-term outcomes of newborns from PPROM pregnancies are presented in Table 4. No differences in the rates of short-term neonatal outcomes were found between colonization and negative amniotic fluid. Colonization was related to a lower compound neonatal morbidity than intra-amniotic infection as per crude analysis, but this was not so after adjusting for gestational age at delivery.

Discussion
The principal findings of this study are as follows: (1) colonization was found in 11% of PPROM pregnancies; (2) Ureaplasma spp. were the most common amniotic fluid microorganisms associated with colonization; however, the burden was lower than that found in women with intra-amniotic infection; (3) the gestational age at rupture of membranes in women with colonization was similar to that of women with negative amniotic fluid but higher than in those with intra-amniotic inflammation; (4) the intensity of intra-amniotic inflammatory response (IL-6 concentrations) was higher in women with colonization than in those with negative amniotic fluid; (5) the rates of acute inflammatory placental lesions was higher in women with colonization than in those with negative amniotic fluid; and (6) short-term neonatal morbidity did not differ between newborns from PPROM with colonization and those with negative amniotic fluid. Microbial invasion of the amniotic cavity represents a condition with two clinical phenotypes: (1) intraamniotic infection when intra-amniotic inflammation is present and (2) colonization/contamination when intraamniotic inflammation is absent 16,19,20 . The rates of these phenotypes might differ among cohorts of women with PPROM due to differences in distribution of gestational age at rupture of membranes, race/ethnicity, and latency interval between ruptured membranes and sampling. In this study, the rates of intra-amniotic infection and colonization were almost equal (12% and 11%, respectively). The rate of colonization found in this study was similar to that reported previously (12%) in the study by Romero et al. 16 ; however, the rate of intra-amniotic infection was lower than that reported previously (12% vs. 29%) 16 . These results show that colonization can be responsible for a significant amount (30-50%) of cases with microbial invasion of the amniotic cavity in PPROM pregnancies. This observation is of clinical significance. However, we must be aware that cases with colonization remain clinically hidden unless the assessment of both microbial invasion of the amniotic cavity and intraamniotic inflammation is employed. This is in direct contrast to cases with intra-amniotic infection (certainly,  Compound neonatal morbidity the need for intubation, and/or respiratory distress syndrome, and/or transient tachypnea of newborns, and/or bronchopulmonary dysplasia, and/or retinopathy of prematurity, and/or intraventricular hemorrhage, and/or necrotizing enterocolitis, and/or intestinal perforation, and/or early-onset sepsis, and/or late-onset sepsis, and/or retinopathy from prematurity, and/or neonatal death before hospital discharge 24 www.nature.com/scientificreports/ along with those with sterile intra-amniotic inflammation), for which a less expensive and less time-consuming assessment of intra-amniotic inflammation is sufficient.
There is solid evidence that Ureaplasma spp. are the most common pathogens in the amniotic fluid from PPROM 16,23 . These low virulent commensal bacteria, commonly found in the vagina and/or cervix are among the smallest self-replicating microorganisms that can grow independently [24][25][26] . In this study, the rate of Ureaplasma spp. in women with colonization reached 60%. In addition, a vast majority of microorganisms, other than Ureaplasma spp., identified in the amniotic fluid from this subset of women, were bacteria commonly present in the vagina, cervix, or rectal niche. These observations support that ascension of the bacteria from the lower genital tract might cause colonization in women with PPROM.
The microbial burden of the amniotic fluid with Ureaplasma spp. varies from hundreds to billions of copies of DNA per milliliter of amniotic fluid in PPROM and it is higher in women with intra-amniotic infection Table 2. Demographical and clinical characteristics of women with preterm prelabor rupture of membranes with respect to the presence of intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity and negative amniotic fluid. Abbreviations: CRP: C-reactive protein, IL: interleukin, WBC: White blood cells. Continuous variables were compared using a nonparametric Kruskal-Wallis or Mann-Whitney U test. Categorical variables were compared using chi-square test or Fisher's exact test. Statistically significant results are marked in bold. Continuous variables are presented as median (interquartile range) and categorical as number (%). p value-comparison among women with intra-amniotic infection, with sterile intra-amniotic infection, with colonization of the amniotic cavity, and with negative amniotic fluid. p value 1comparison between women with colonization of the amniotic cavity and with intra-amniotic infection. p value 2 -comparison between women with colonization of the amniotic cavity and with sterile intra-amniotic inflammation. p value 3 -comparison between women with colonization of the amniotic cavity and with negative amniotic fluid.

Intra-amniotic infection (n = 37)
Sterile intra-amniotic inflammation (n = 21)    www.nature.com/scientificreports/ than in those with colonization 21 . Accordingly, a lower burden of Ureaplasma spp. was observed in women with colonization than in those with intra-amniotic infection in this study. Given the dose-dependent relationship between intra-amniotic inflammatory response (measured by IL-6 concentrations) and the amniotic fluid burden of Ureaplasma spp. in PPROM pregnancies 21,27,28 , it is likely that women with colonization, caused by Ureaplasma spp., could not elicit an intra-amniotic inflammatory response intense enough to pass a clinical threshold for intra-amniotic inflammation. In addition, clinicians should be aware of the following facts: (1) intra-amniotic inflammation is expressed as a categorical condition (present/absent) and (2) IL-6 is a physiological constituent of amniotic fluid (measurable concentrations of IL-6 in all PPROM pregnancies. Collectively, distinguishing between the early-stage of the "classical" intra-amniotic inflammatory response to a microorganism with a weak inflammatory response and just the presence of microorganisms in the amniotic fluid with the absence of an intra-amniotic inflammatory response is at this stage almost impossible. There is evidence that the rate of intra-amniotic infection in PPROM pregnancies decreases with advanced gestational age 16,21 . However, colonization has been previously described only in the subset of PPROM with gestational ages between 25 and 33 weeks 16 . In contrast, colonization in PPROM was found in this study only beyond the 30th week of gestation. In addition, the range of gestational ages at rupture of membranes among women with colonization was very narrow (only 46 days) compared to those with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid, in which the ranges were almost two-fold higher. These findings might extend our previous observation, where an intensive intra-amniotic inflammatory response to amniotic fluid bacteria, characterized by concentrations of multiple inflammatory-related proteins in amniotic fluid, was not found beyond gestational 32 weeks 29 .
The intra-amniotic inflammatory response is associated with elevated concentrations of various cytokines, chemokines, and other inflammation-related proteins and lipids in the amniotic fluid 30,31 . This condition is typically followed by the attraction of neutrophils towards the amniotic cavity from the intervillous space of the placenta into the chorionic plate and/or from the decidua into fetal membranes, leading to the development of acute inflammatory lesions in the placenta 32 . In this study, both intra-amniotic inflammatory responses, measured by concentrations of IL-6 in the amniotic fluid and the presence of acute inflammatory placental lesions, were assessed. Collectively, women with colonization had a higher intensity of intra-amniotic inflammatory response and rates of acute inflammatory placental lesions than women with negative amniotic fluid. This observation further supports the hypothesis mentioned above that colonization in PPROM represents an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation. www.nature.com/scientificreports/ It remains debatable whether the microbial invasion of the amniotic cavity and/or intra-amniotic inflammation, after correction for gestational age at delivery, is associated with worse neonatal outcomes 16,21,33 . To extend this knowledge, the selected aspects of short-term neonatal morbidity were investigated in this study. After adjusting for gestational age at delivery, no differences were found between newborns from PPROM with colonization and negative amniotic fluid.
This study had several strengths. First, a relatively large cohort of women with singleton pregnancies complicated by a well-defined clinical phenotype of spontaneous preterm labor (PPROM) with available information about the intra-amniotic environment and histopathology of the placenta was used. Second, a thorough assessment of microbial invasion of the amniotic cavity consisting of specific PCR for Ureaplasma spp., M. hominis, and Ch. Trachomatis; non-specific PCR for 16S rRNA; and aerobic/anaerobic cultivation were employed in this study. Finally, short-term neonatal outcomes were available for all newborns in the PPROM cohort.
This study also has limitations that are worth mentioning. First, this study consisted of a homogeneous population of Caucasian women living in the eastern part of the Czech Republic. This prevents the findings of this study from being generalized to populations with racial/ethnic disparities. Second, only loads of Ureaplasma spp. DNA, but not all bacterial DNA, in the amniotic fluid were evaluated in this study. This shortcoming prevented us from assessing whether the burdens of amniotic fluid bacteria other than Ureaplasma spp. differ between women with colonization and those with intra-amniotic inflammation. However, in a recent study on women with preterm labor with intact membranes, microbial and fungal burdens in the amniotic fluid were lower in those with microbial invasion of the amniotic cavity without intra-amniotic inflammation than in those with intraamniotic inflammation 17 . Last, the long-term outcomes of infants from PPROM pregnancies were not available.
In conclusion, colonization in PPROM, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation.

Methods
This retrospective study included all pregnant women admitted to the Department of Obstetrics and Gynecology of the University Hospital Hradec Kralove, Czech Republic, between December 2018 and July 2021, who met the following criteria: (1) age ≥ 18 years, (2) PPROM between gestational ages 24 + 0 and 36 + 6 weeks, (3) singleton pregnancy, and (4) transabdominal amniocentesis. The exclusion criteria were as follows: (1) pregnancy-related and other medical complications (e.g., pregestational diabetes, gestational hypertension, and preeclampsia), (2) congenital or chromosomal fetal abnormalities, (3) signs of fetal hypoxia, and (4) significant vaginal bleeding.  Maternal blood and amniotic fluid samples were obtained at the time of admission before the administration of corticosteroids, antibiotics, or tocolytics. The performance of transabdominal amniocentesis to assess the intra-amniotic environment has been a part of the department's standard clinical management of women with PPROM. Women with PPROM were further managed based on amniotic fluid test results. Those with confirmed intra-amniotic inflammation received intravenous clarithromycin for seven days unless delivery occurred. Women without intra-amniotic inflammation were treated with intravenous benzylpenicillin (intravenous clindamycin in case of penicillin allergy) for seven days unless delivery occurred. Once the final results regarding microbial invasion of the amniotic cavity from cultivation and/or PCR were known, the attending clinician decided to modify the antibiotic therapy. Corticosteroids (betamethasone) were administered to those at gestational ages between 24 + 0 and 34 + 6 weeks to accelerate lung maturation. Tocolysis (atosiban) was used in those who developed regular uterine activity during the course of corticosteroid therapy or within 24 h after their administration. Table 4. Short-term neonatal morbidity of newborns from pregnancies with PPROM with respect to intraamniotic infection, sterile intra-amniotic inflammation, colonization, and negative amniotic fluid. Variables were compared using the Fisher's exact test and are presented as number (%). Statistically significant results are marked in bold. p-value 1 -comparison between women with colonization of the amniotic cavity and with intra-amniotic infection. p-value 2 -comparison between women with colonization of the amniotic cavity and with sterile intra-amniotic inflammation. p-value 3 -comparison between women with colonization of the amniotic cavity and with negative amniotic fluid. # the results were adjusted for gestational age at sampling.    46 . Colonization was defined as the presence microbial invasion of the amniotic cavity without intraamniotic inflammation. Intra-amniotic infection was defined as the concurrent presence of microbial invasion of the amniotic cavity and intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without microbial invasion of the amniotic cavity. Negative amniotic fluid was defined as amniotic fluid without intra-amniotic inflammation and microbial invasion of the amniotic cavity. The intra-amniotic inflammatory response was characterized by the concentration of IL-6 in the amniotic fluid. Maternal inflammatory response was determined by the concentrations of CRP concentrations and WBC counts in maternal blood. HCA was diagnosed based on the histological grades 3-4 for the chorion-decidua, and/or grades 3-4 for the chorionic plate, and/or grades 1-4 for the umbilical cord, and/or grades 1-4 for the amnion 44 . Funisitis was diagnosed based on histological grades 1-4 for the umbilical cord 44 . Inflammation of the amnion was diagnosed based on histological grades 1-4 for the amnion 44 . Compound neonatal morbidity was defined as the need for intubation, and/or respiratory distress syndrome, and/or transient tachypnea of newborns, and/or bronchopulmonary dysplasia, and/or retinopathy of prematurity, and/or intraventricular hemorrhage, and/or necrotizing enterocolitis, and/or intestinal perforation, and/or early-onset sepsis, and/or late-onset sepsis, and/or neonatal death before hospital discharge 20 .
Statistical analyses. The demographic and clinical characteristics of the patients were compared using the non-parametric Kruskal-Wallis or Mann-Whitney U tests for continuous variables, as appropriate, and Fisher's exact or chi-square tests for categorical variables, as appropriate, and results were presented as median (interquartile range [IQR]) and number (%), respectively. The normality of the data was tested using the Anderson-Darling test. The loads of Ureaplasma spp. DNA in the amniotic fluid were not normally distributed; therefore, the non-parametric Mann-Whitney U test was used for the analyses. Kaplan-Meier survival curves were constructed, and a log-rank (Mantel-Cox) test was used to compare survival distributions among women with colonization, sterile intra-amniotic inflammation, and negative amniotic fluid. Spearman's partial correlation was used to adjust the results for gestational age at delivery. Differences were considered significant at P < 0.05. All p-values were determined using two-tailed tests, and all statistical analyses were performed using GraphPad Prism v8 for Mac OS X (GraphPad Software, San Diego, CA, USA) and the Statistical Package for the Social Sciences (SPSS), version 28.0.0.0, for Windows (SPSS Inc., Chicago, IL, USA).